Trypanosoma cruzi is an obligate intracellular parasite that causes Chagas'disease, a chronic and debilitating infection that affects millions of people. Approximately 25% of the population in Latin America is at risk of acquiring this infection. Clinical reports of Chagas'disease, mostly of immigrants from endemic countries, are increasing in the US. Blood banks in the US are required to screen for potential chagasic donors. Cardiomyopathy is the most common expression of the infection and sudden cardiac death accounts for 55-65% of deaths. Nifurtimox and Benznidazole are the only chemotherapy available. In the US, these drugs can only be obtained through the CDC. Chemotherapy is recommended in the acute phase and during Chagas'reactivation in immunosuppressed or HIV infected patients. Treatment can achieve parasitological cure during the acute phase but has little efficacy during the chronic phase of infection, and patients require careful monitoring due to severe adverse effects. New drugs with less toxicity and higher parasitological cure rates are essential. The goals of this project are to i) develop, optimize and validate an Image-Based High Throughput Drug Screening (HTS) Assay for the intracellular stages of T. cruzi, and ii) identify selective inhibitors of T. cruzi proliferation. The HTS assay allows the evaluation of effective compounds with both laboratory and field isolates reflecting the parasite genetic diversity. Different host cells including macrophages and cardiomyocytes can also be used to better model human infection. The automated fluorescence microscope based HTS assay provides a powerful tool to analyze the effect of compounds at the level of single cells and determine cytotoxicity levels for parasites and host cells. A Z-factor of 0.45 was achieved in preliminary studies. Optimization of the HTS assay will first aim at improving the Z-factor, followed by switching from a manual to a fully automated system using our in-house robot dispenser instrument. The HTS assay will constitute an important advancement in drug discovery efforts for Chagas'disease. PUBLIC HEALTH RELEVANCE: Our aim is to develop, optimize, and validate a cell-culture, microscopy-based HTS assay for the pathogenic intracellular stage of T. cruzi, and to screen compound libraries from industrial and academic sources. This HTS assay will greatly facilitate drug discovery efforts for Chagas'disease.